Stages Of Endothelial Dysfunction In Atherosclerosis Biology Essay

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Atherosclerosis is a medical term derives from Greek, athere meaning gruel that soft consistency of the core of the plaque (mainly lipid), and sclerosis, meaning thickening or hardening in body artery. Cholesterol of low-density lipoprotein (LDL) particles is mainly lipid of atherosclerotic plaque that has left the circulation [1,4]. Atherosclerosis is an inflammatory disease, which featured by complex accumulation of lipids and rich plaques in the inner layer of the wall of an artery which is known as Intima [2].

Plaque is made of cholesterol, phospholipids, live and dead cells, cholesterol esters and fibrin [3]. Figure 1.1 illustrates the difference between: normal human artery (Left) abnormal artery filled with plaques (Right).

Figure 1.1 illustrates the difference between: normal human artery (Left) artery plaques (Right) (Britannica, 2007).

Now, chronic inflammation is seen as a stamp of atherosclerosis (AH), where an increase in inflammatory mediators donates towards the initiation, progression, and rupture of atherosclerotic plaques. Also, the development of an atherosclerotic plaque depends on an interaction of the cellular components of the immune system as cytokines, monocytes, and cell adhesion molecules (CAM). The significance of inflammation and of the monocyte derived macrophage in atherosclerosis is widely recognised with macrophages' being the predominant leukocyte present in the atherosclerotic lesions [1,4].

Atherosclerosis involves multiple processes. The early-stage of atherosclerotic lesions is the fatty streak and there are three key stages of atherogenesis: a) Initiation, which is composed of lipid -loaded macrophages known as foam cells, that endothelial dysfunction, increased penetration and adhesiveness to leukocytes. Accumulation of lipids by arterial tissue macrophage and pro-coagulation factors are released b) Progression, is a chronic inflammation caused by cytokines and mitogenic chemotactic factors. Secretion of collagen and proteoglycan. c) Complication, leading to plaque rupture and thrombosis [4,5]. Figure 1.2 illustrates Stages of endothelial dysfunction in atherosclerosis.

Figure 1.2 Stages of endothelial dysfunction in atherosclerosis (Grahams, 2007).

Histologically, it has been demonstrated that atherogenic lipoproteins similar as oxidized (LDL), remnant lipoprotein (β-VLDL) and lipoproteins (LP) are play a crucial role in the pro-inflammatory reaction, whereas (HDL), anti-atherogenic lipoproteins, used anti-inflammatory functions. Atherosclerosis known as a major involvement of diabetes and it leads to death in most of diabetes cases. Diabetes is an epidemic affects >250 million world wide and at least 10% of hospital beds are occupied by diabetes patients. There are three majors mechanisms enclose most of pathological changes observed in accelerated atherosclerosis a) Non-enzymatic glycosylation of lipids and proteins. b) Oxidative stress c) protein kinase C (PKC) activation [5,6].

Well, is required to completely clarify further research in atherosclerosis and inflammation there is much increasing evidence that clear subpopulations of monocytes that can differentiate into specific macrophage subpopulations in respond to cytokine stimulus [ 4 ].

1.1.1 Risk factors for atherosclerosis

Atherosclerosis is a multifactorial disease including of both genetic and environmental factors which all leading to the predisposition of atherosclerosis complications. Most risk factors can be controlled and atherosclerosis can be prevented and delayed. A number of risk factors have been identified: hyperlipidaemia, hypertension, diabetes, smoking, obesity and lack of physical activity. Also, the risk factors which can't control with atherosclerosis are age and family history of early heart disease.

Hyperlipidaemia represents an essential risk factor for atherosclerosis that is by alteration in the lipid profile plasma, including increase total cholesterol, triglycerides, and low density lipoprotein (LDL) levels as well as decrease in high density lipoprotein (HDL) levels[7].

A strong genetic studies to atherosclerosis has been reported, with familial hypercholesterolemia or experimental (transgenic animals, specifically those lacking the LDL receptor or apolipoprotein E genes), confirmed the relationship between abnormal cholesterol metabolism and atherosclerosis.

Cigarette smoking is a strong correlation to cause atherosclerosis morbidity and mortality, also smoking increase inflammation, thrombosis and oxidative stress.

Hyperglycaemia is now proven as the primary cause of accelerated atherosclerosis in diabetes. There are a number of processes that leading to atherogenesis in type 2 diabetes (T2D) patients which have been identified:

1- Increased in the formation of advanced glycosylation end-product (AGEs), some may cause to the production of auto antibodies. In most population increase obesity has been associated with classical risk factors for atherosclerosis and T2D.

2- Diabetes patient may have an increased degree of microinflammation characterized by increased levels of C-reactive protein (CRP).

3- Coagulation abnormalities, mainly increased concentration of plasma fibrinogen and platelet abnormalities have also been described.

4- Typical cluster of abdominal obesity, hyperinsulinaemia, insulin resistance and dyslipidaemia also includes hypertension.

Obesity and lack exercise are main factors which cause of preventable death, a growing epidemic, and a major development diabetes, hypertension and CVD. Increased consumed of fat diet is related to the frequency of dyslipidaemia. Also, the spiralling global epidemic of obesity is related to the development of metabolic abnormalities, such as the metabolic syndrome associated with insulin resistance and decrease HDL[ 8].

1.2 Macrophage and Monocyte

Macrophages / Monocytes are cells derived from myelomonocytic stem cell in bone marrow. Actually, monocytes and macrophages were classified as cells of the reticulo-endothelial system - RES (Aschoff, 1924). Van Furth et al. (1972) proposed the mononuclear phagocyte system -- MPS, and monocytes and macrophages became basic cell types of this system. Their development start in the bone marrow and passes by the following steps: stem cell - committed stem cell - monoblast - promonocyte - monocyte (bone marrow) - monocyte (peripheral blood) - macrophage (tissues) [4].

Monocytes circulate with a half-life of 1-3 days and then move to various tissues and start collectively which known as Macrophage. The role of macrophages and monocytes has a well-established in inflammatory process and this role depend on their capacity to produce free oxygen radicals, proteases, complement factors and cytokine [4,9].

There are two types of macrophages, normal and inflammatory macrophages. Normal macrophage include connective tissue ( histocytes), liver (kupffers cells), lung (alveolar macrophages), lymph nodes (free and fixed macrophages), bone marrow (fixed macrophages), skin (histiocytes, Langerhans cell) and in other tissues.

Inflammatory macrophages can be found in various exudates. They may be used by different special markers, e.g. peroxidase activity, and they are derived exclusively from monocytes they share similar prosperities. The term exudate macrophages designate the developmental stage and not the functional state [ 9].

The monocyte / macrophage are phagocytic cells that are crucial for tissue homeostasis and affect all tissues. Tissue macrophages migrate into the tissues in response to inflammatory signals [10].

Macrophage has been identified in atherosclerotic plaque in the 1960 [11]. New insights into the essential of inflammatory cells monocyte and macrophage that are involved in all stages of atherosclerosis pathogenesis [1,12,13].Figure 1.3 illustrate monocyte/macrophage in atherosclerotic plaque.

(Prakash, 2009)

Figure 1.3 Monocyte/macrophage in atherosclerotic plaque. (1) Endothelial dysfunction (pink cells) leads to the up regulation of vascular adhesion molecules (Pselectin, ICAM and VCAM) that along with the binding of MCP-1 to its receptor CCR2 facilitate monocyte activation and adhesion to the endothelium. (2) LDL is taken up by the endothelium and is converted to mm-LDL and Ox-LDL by the action of ROS. Having migrated across the endothelium, monocytes differentiate into macrophages in the presence of factors such as MCSF. (3) These macrophages accumulate Ox-LDL through scavenger receptors (CD36 and SR-A) and form foam cells. (4) Interaction of foam cells with T cells leads to the production of inflammatory mediators that promote the migration of smooth muscle cells into the intima.

1.3 M1 and M2 monocytes

Two types of monocytes with clear patterns of surface markers and behaviours through inflammation have currently been characterized, M1 (pro-inflammatory) and M2 (anti-inflammatory). Several of macrophage phenotypes derived from these monocytes subsets in response to mediators of innate and acquired immunity have also been found in plaques, classically activated M1 and alternatively activated M2 macrophages. While Th1 cytokines, such interferon gamma (INFγ), IL1-β and lipopolysaccharide (LPS), induce a "classical" activation profile (M1), Th2 cytokines, an example as IL-4 and IL-13, and induce an "alternative" activation program (M2) in macrophages [4,14].

M1 macrophages are potent effectors cells that kill microorganisms and produce primarily pro-inflammatory cytokines, including TNFα, IL-6 and IL-12 (Gordon, 2003). Figure 1.4 shows the hypothetic mechanism of action between T-helper lymphocytes, cytokines, inflammatory mediators and PPARγ for the generation of monocyte lineage to the M1 or M2 phenotype [ 14].

Th1 cytokines (IFNγ, IL1β) Th2 cytokines

and bacteria LPS (IL-13, IL-4)

M1 Macrophage M2 Macrophage


Down Regulation Up Regulation

M1 Markers M2 Markers

(TNFα, MCP-1, IL-6, IL-12) (MR, CD163, AMAC, TGFβ, IL-1β

Receptor antagonist, IL-10)

Figure 1.4: Monocytes/Macrophage heterogeneity; Monocytes under the influence of Th2 cytokines IL-13/IL-4 or inflammatory mediators such as IFNγ, IL-1β or LPS are polarised through the influence of PPARγ activation into M2 or M1 mature macrophages respectively.

1.4 Mannose Receptor (MR)

The mannose receptor (MR; CD206) is a member of the calcium-dependant lectin receptor (CLR) family that have characteristic carbohydrate recognition domains with selective binding to specific glycan's. MR is expressed firstly by tissue macrophages, lymphatic, hepatic and endothelial cells in humans and mice. MR role in carbohydrate pattern recognition, its effective capacity of endocytosis, and its play role in phagocytosis of microorganisms, MR have been suggested to play a dual role in host defence and homeostasis. The role of MR in homeostasis of serum glycoprotein has been conclusively demonstrated through the development of MR-deficient mice [14].

The biology of MR has given new insights of its use as a target for delivering drugs to macrophages that have internalized bacteria or other infectious organisms [15].

1.5 Peroxisome Proliferators-activated receptors

Peroxisome Proliferators-activated receptors (PPARs) are family group members of the nuclear receptor proteins that function as transcription factors which are regulating the expression of genes and include retinoic X receptor (RXR), thyroid hormone receptor, and vitamin D receptor. PPARs subfamily have a similar structure including C region with twin zinc finger DNA binding domain, N-terminal A/B region with AF1 ligand-independent transactivation domain, AF2 ligand-dependent transactivation domain at the extreme C terminus and D,E and F regions with a large and flexible ligand binding site [16].

PPAR play an essential role in the regulation of cellular differentiation, metabolism (carbohydrate-protein-lipid) development and tumorigenesis of organisms. PPARs are activated by nutrient molecules and their derived. Increasing level of PPARs has been recognised as an important role in metabolic disorders, hypertension and cardiovascular disease (CVD) [ 16].

There are three PPAR isoforms, encoded on separate genes have been identified: PPARα (Alpha), PPARβ or δ (Beta or Delta) and PPARγ (Gamma).PPARs now have been recognized as an essential of macrophage polarisation Figure 1.5 illustrates PPAR isoforms. They stimulate various effects on intracellular and extracellular lipid metabolism, cell proliferation, glucose homeostasis, and control of inflammation and arteriosclerosis [17].

PPARs have received a significant attention from researches due to their many potential roles and involvement in pathological states including atherosclerosis, diabetes, inflammation, obesity, cancer, infertility, wound healing and the design new drugs [ 16].

Figure 1.5 illustrates PPAR isoforms. Adopted from (Jung, 2004)

1.5.1 PPARα

Mostly expressed in liver, kidney, intestine, and heart also, increases the production of enzymes involved in fatty acid oxidation. PPARα activators contain the fatty acids and the fibrate class of hypolipidemic drugs and fatty acids this receptor sub-type is required for peroxisome proliferation in a rodent liver Figure 1.6 illustrates PPAR alpha pathway [16].

©2009 Jack Vanden Heuvel, Penn State University

Figure 1.6 illustrates PPAR alpha pathway.

1.5.2 PPARδ and PPARβ

PPARδ is a sub-type expressed in many tissues, but mainly in brain, skin, adipose tissue and macrophage. PPAR beta/delta; activation leading increase lipid catabolism in skeletal muscle, heart and adipose tissue and improves the serum lipid profile and insulin sensitivity in many animal models. Also, PPAR beta/delta; ligands prevent weight obtain and suppress macrophage-derived inflammation. These data are hopeful and indicate that PPAR beta/delta; ligands may become a therapeutic option for the treatment of metabolic syndrome [18]. Skeletal muscle insulin resistance is a primary risk factor for the development of T2D. There is high evidence that PPARδ is an essential regulator of skeletal muscle metabolism, in particular, muscle lipid oxidation, highlighting the potential benefits of this isoform as a drug target. PPARδ seems to be a key control of skeletal muscle fibre type and a possible mediator of the adaptations noted in skeletal muscle in response to exercise [ 19].

1.5.3 PPARγ

PPARγ mainly expressed in adipose tissue also, in macrophages, heart, colon and other tissues. PPAR gamma is working as a transcription factor after it heterodimerizes RXR and binds to specific response elements. In addition is a ligand-dependent transcription factor that controls the expression of specific target genes involved in adipogenesis, inflammatory responses and lipid metabolism. PPAR gamma is a member of this gene family that is activated by fatty acids and thiazolidinedione drugs (TZDs) that plays a role in insulin sensitivity and adipogenesis Figure 1.7 PPAR gamma pathways [20].

Figure 1.7 PPAR gamma pathways.

Synthetic ligands for PPARγ include the anti-diabetic insulin sensitizer's thiazolidinediones (TZDs), such as pioglitazone, rosiglitazone and troglitazone. PPARγ agonists was shown to enhance the expression of PPARγ in macrophages and subsequently inhibit synthesis of (SR-A), partly through the inhibition of NF-κB. PPARγ dependent inhibition of NF-κB has also been implicated in the anti-inflammatory inhibition of key cytokines, contain TNFα and IL-6 [20,21]. Also, was suggested the natural PPARγ agonists found in foods may be benefit to human health by acting as anti-inflammatory molecules and more interest to the food industry [22]. Another researched related to PPARγ activation not only suppresses osteoblastogenesis, but also activates osteoclastogenesis, leading to decreasing bone formation while sustaining or increasing bone resorption. The pro-osteoclastogenic effect of rosiglitazone is mediated by a transcriptional network comprised of PPARγ, PPAR-gamma coactivator 1β and estrogen-related receptor α, which promotes both osteoclast differentiation and mitochondrial activation. So, PPARγ plays two roles in bone homeostasis by regulating both mesenchymal and hematopoietic lineages [23].

1.6 Conjugated Linoleic Acid (CLA)

Conjugated linoleic acid (CLA) is a class of 18-carbon polyunsaturated fatty acids and massive found in the meat of ruminant animals and dairy products. CLA is an octadecenoic acid (18:2) with tow conjugated double bonds, predominantly found in the cis 9 and trans 11 or trans 10 and cis 12 positions The presence of a conjugated double bonds meaning that there are two types of configurations, one in which the same substituent's of the carbon atoms forming the double bond are located on the same side with respect to the plane that determines the carbon-carbon double bond, is cis, or on opposite sides, trans form. Figure 1.8 illustrates the shape of cis and trans [24].

Cis Trans

Figure 1.8 chemical forms of cis and trans.

In addition conjugation of double bonds take place as part of free radical-mediated oxidation of LA, CLA is a true isomer of LA that does not have additional oxygen [25]. Figure 1.9 illustrates Isomer structure of CLA.

Ordinary LA, c-9, c-12-octadecadienoic acid

t-9, c-11-CLA

t-10, c-12-CLA

COOH end

Figure 1.9 illustrates Isomer structure of t-10, c-12-CLA (top), c-9, t-11-CLA (centre), and ordinary LA, c-9, c-12-octadecadienoic acid (bottom). The molecules are aligned at their carboxyl end to show the double bonds (yellow) on molecular shape (Adopted from: Steinhart, 2006).

CLA has been demonstrated to have anti-diabetic, anti-atherosclerotic and anti-carcinogenic effects in cell lines and animal models [26]. Also, was found to have a strong immune modulating activity described by increased blastogenesis and macrophage killing ability. CLA is formed through the microbial process of fermentative bacteria as result of defective bio-hydrogenation (BH) of dietary fatty acids which are quickly hydrolyzed into free unsaturated fatty acids. Food products derive from these animals such as meat, milk, and dairy products are the major sources of CLA in the human diet [27]. Figure 1.10 Predominant pathways of bio-hydrogenation of unsaturated C18 fatty acids.

c-6, c-9, c12 c-9, c-12 c-9, c-12, c-15

(γ linoleic acid) (Linoleic acid) (α linoleic acid)

c-6, c-9, t-11 c-9, t-11 c-9,t-11,c-15

(Conj. octadecatrienoic acid) (Conj. octadecatrienoic acid) (Conj. octadecatrienoic acid)

c-6, t-11 t-11, c-15

(Octadecadienoic acid) (Octadecadienoic acid)


(Vaccenic acid)


(Stearic acid)

Figure 1.10 Predominant pathways of bio-hydrogenation of unsaturated C18 fatty acids.

Diets enriched in linoleic acid have shown to be beneficial in reducing CHD risk in human subjects [28, 29] and have been shown to protect against the development of coronary artery atherosclerosis in monkeys [30] and aortic atherosclerosis in transgenic mice [31]. Work done in hamsters [32] has suggested a potential mechanism for the beneficial effect of dietary linoleic acid in lowering LDL cholesterol concentrations [33].

The heart disease and circulatory system cardiovascular disease (CVD) continue to be the main affected of mortality and morbidity in the UK about 38% recorded in 2003 [34].

The concentration of CLA isomers in dairy products or beef vary depending on the diet offered to cows or steers. The animal-to-animal is variation, animal that grass-fed, produce higher amounts of CLA in their milk than those which receive conserved feed, such as grain, hay and silage. The concentration of CLA in ruminant-derived products ranges from 3 to 8mg CLA/g fat. Cheese products were found to have significantly higher CLA content, range from 3.59 - 7.96 mg/g of lipid while other fermented dairy products contain 3.82-4.66 mg of CLA per gram of lipid [35].

Dietary CLA is being investigated for beneficial effects for disease prevention and treatment in the experimental models types, containing Obesity, T2D, and Cardiovascular Disease [36]. Inflammatory response is the most important period that controls the entire healing process. It was hypothesized that dietary CLA supplementation accelerates cutaneous wound healing by regulating antioxidant and anti-inflammatory functions [37].

1.7- THP-1 cell Line

The human monocytic cell line THP-1 is a promonocytic leukaemia which has isolated from the peripheral blood of a 1 year old patient suffering from the acute monocytic leukaemia in 1980 and it is widely used in cell culture in around the world [38].

THP-1 cells grow in suspension but can be differentiated into macrophage like cells when they are treated with Phorpol 12-myristate 13-acetate (PMA) or vitamin D3 Figure 1.11 illustrate the effects PMA in THP-1 cell.


Undifferentiated Differentiated

Figure 1.11(A) Undifferentiated THP-1 cells, and (B) Differentiated THP-1 cells with PMA.

THP-1 cells can differentiate into macrophage like cells which are mimic native monocyte -derived macrophage in various respects. This cell line is commonly used in the study of foam cell formation in atherosclerosis inflammation. THP-1 express Fc and C3b receptors, but lack with both surface and cytoplasmic immunoglobulin's [38].

Konopka and Duzuges they were investigate to determine whether variability observed in the susceptible of THP-1 cells to HIV-1 infection may be related to the differential expression of CD4, CCR5 and CXCR. It was established that the cell surface CD4 play a primary role in determining how efficiently THP - 1 cells can be infected with X4 and R5 HIV-1 isolate [39].

A previous study was indicated that tissue microenvironments influence HIV-1 replication [40]. Macrophages are early targets of human immunodeficiency virus type -1 (HIV-1) infection and serve as potential reservoirs for long term infection [41]

1.8 Flow Cytometry Analyser (FACS)

Flow cytometry technique, was a first appeared in the sixties of the last century for counting cell populations in suspension. In addition is one of the most methods widely used for analysis of cell sorting, intracellular protein analysis, cell cycle studies, apoptosis analyses and immunophenotyping of acute leukaemia and lymphoproliferative syndromes. FACS analyser has three main systems the sample injection system, optical system, and the electronic informatics (computer) system [42].

Today, AIDS is the main cause of patient's deaths in people aged between 18 and 45, FACS it is interesting play role in monitoring of lymphocyte subsets in AIDS. FACS is a powerful technique, currently is routinely used in many laboratories. FACS performs this analysis through passing thousands of cells per second by a laser beam and capturing the light that emerges from each cell as it passes through. Then the data collect to analyse statistically by FACS software to report cellular characteristics such as size, complexity, phenotype and health [43].

1.9 AIM